CN117653587A

CN117653587A - Topical external preparation containing pirfenidone and application thereof

Info

Publication number: CN117653587A
Application number: CN202311138820.2A
Authority: CN
Inventors: 刘飞; 姜伟化; 石莉; 李培冉; 曹晓琴; 杜子蝶; 王婕; 张翠霞
Original assignee: Nanjing Minoway Medical Technology Co ltd
Current assignee: Nanjing Minoway Medical Technology Co ltd
Priority date: 2022-09-06
Filing date: 2023-09-05
Publication date: 2024-03-08
Also published as: WO2024051687A1

Abstract

The present invention provides a topical external preparation of pirfenidone comprising a therapeutically effective amount of pirfenidone, water, and at least one co-solvent selected from the group consisting of dimethyl sulfoxide and an alcoholic solvent. The preparation has high skin permeability, good stability, no irritation, good skin tolerance, and convenient use, and can be used for preparing medicine for preventing or treating skin or connective tissue hyperplasia related diseases and chronic skin injury.

Description

Topical external preparation containing pirfenidone and application thereof

This application claims priority from the following prior applications: patent application number 202211086265.9 filed by 2022, 09 and 06 to China national intellectual property agency is a prior application with the name of 'a topical external preparation containing pirfenidone and application thereof'. The entirety of the prior application is incorporated by reference into this application.

Technical Field

The invention belongs to the field of pharmaceutical preparations, and in particular relates to a local external preparation containing pirfenidone and application thereof.

Background

Pirfenidone (pirfenidone), chemical name 5-methyl-1-phenyl-2- (1H) pyridone, is a compound with broad spectrum anti-fibrosis effect, can prevent and reverse fibrosis and scar formation, and has the chemical structure shown as follows:

in 2014, pirfenidone was approved by the U.S. Food and Drug Administration (FDA) for marketing under the trade nameCurrently, pirfenidone has become the first drug approved in several countries for the treatment of Idiopathic Pulmonary Fibrosis (IPF).

In recent years, there have been extensive studies on other uses of pirfenidone. For example, WO00/16775 discloses the use of pirfenidone for the treatment and prophylaxis of dermatological lesions, in particular lesions of fibrotic nature, such as fibrotic damaged tissue, contagious warts, contact dermatitis, burns and scars, etc. WO99/47140 discloses the use of 2-1H-pyridones, in particular pirfenidone, as topical disinfection treatments for the treatment of fungal, bacterial and the like, on the surface of the skin. WO01/62253 discloses the use of pirfenidone for the treatment of epilepsy. Pirfenidone is disclosed in US patent 4,052,509 as having use as an anti-inflammatory agent. Pirfenidone has anti-fibrotic activity as disclosed in mexico patent 182,266. Chinese patent document CN111246840a discloses that pirfenidone can be used for the prevention or treatment of chronic skin lesions and lesions caused by neuropathic ulcers, in particular for the treatment of diabetic foot syndrome and for the treatment of vascular ulcers.

In summary, many studies have shown that pirfenidone is useful for the treatment and prevention of skin lesions such as surgical wounds, burns, accidents, ultraviolet rays, fibrosis or inflammatory skin abnormal scars and other defects caused by pregnancy or chemical agents, but pirfenidone is poorly soluble in water and is difficult to prepare into stable external preparations.

CN10808641a discloses a gel composition containing pirfenidone, which discloses that a solubilizing agent such as NMP, ethanol and propylene glycol is selected to prepare a gel preparation, which can be used for tissue restoration with fibrotic lesions and prevention of fibrotic lesions, but stability, transdermal effect, in vivo effect and the like thereof are not studied.

Therefore, there is a need to further provide a pirfenidone pharmaceutical composition with high stability, good transdermal effect and therapeutic effect.

Disclosure of Invention

The invention aims to provide a pirfenidone topical external preparation which has high skin permeability, good stability and low irritation, can permeate the skin efficiently and is safe to take effect.

In order to achieve the above purpose, the present invention provides the following technical solutions:

in a first aspect, the present invention provides a topical external preparation of pirfenidone comprising a therapeutically effective amount of pirfenidone, water, and at least one co-solvent.

In some embodiments, the co-solvent is selected from the group consisting of Dimethylsulfoxide (DMSO) and an alcoholic solvent selected from one, two or more of ethanol, propylene glycol, isopropanol, 1-propanol, butylene glycol. Preferably, the cosolvent is a combination of DMSO and ethanol, or a combination of DMSO and propylene glycol, or a combination of DMSO, ethanol and propylene glycol.

In some embodiments, the amount of water in the formulation is from 10wt% to 55wt%, preferably from 15wt% to 50wt%, for example 20wt%,25wt%,30wt%,35wt%,40wt% or 45wt%.

In some embodiments, when the co-solvent is a combination of DMSO and ethanol, the formulation comprises 15wt% to 35wt% DMSO, 15wt% to 30wt% ethanol, based on the total weight of the topical formulation; preferably, the formulation comprises 15wt% to 25wt% DMSO, 15wt% to 25wt% ethanol, for example containing 20wt% DMSO and 20wt% ethanol, or containing 20wt% DMSO and 25wt% ethanol.

In some embodiments, when the co-solvent is a combination of DMSO, ethanol, and propylene glycol, the formulation comprises 15wt% to 35wt% DMSO, 15wt% to 30wt% ethanol, 15wt% to 25wt% propylene glycol, based on the total weight of the topical formulation; preferably, the formulation comprises 15wt% to 25wt% DMSO, 15wt% to 25wt% ethanol, and 20wt% to 25wt% propylene glycol.

In some embodiments, the pirfenidone in the formulation is present in an amount of 1wt% to 14wt%, based on the total weight of the pirfenidone topical external formulation; preferably, present in an amount of 4wt% to 12 wt%; for example in an amount of 6wt% to 12wt%, for example in an amount of 7wt%, 8wt%, 9wt%, 10wt%, 11 wt% or any interval range thereof.

In some embodiments, the topical pirfenidone formulations of the present invention may be formulated into different dosage forms, such as solutions, suspensions, creams, ointments, lotions, and gels. Preferably, the pirfenidone topical external preparation of the present invention is a solution or gel. More preferably, the pirfenidone topical external preparation of the present invention is a gel.

In some embodiments, optionally, the pirfenidone topical external preparation further comprises a gelling agent. The gelling agent may be one known in the art for topical external preparations including, but not limited to: one, two or more of cellulose derivatives, polyvinylpyrrolidone, carbomer polymers, carbomer derivatives, maltodextrin, polydextrose, dextrates, polyacrylamide gellants, carboxypolymethylenes, polyvinyl alcohols, poloxamers, polyethylene glycols; preferably, the gelling agent is selected from one, two or more of cellulose derivatives, carbomer polymers, carbomer derivatives. For example, the gelling agent is HPC, carbomer 980, carbomer 981, and the like.

In some embodiments, the gelling agent is present in an amount of 0.05wt% to 5wt%, preferably in an amount of 0.1wt% to 2wt%, for example 0.3wt% to 1wt%, based on the total weight of the pirfenidone topical external preparation.

In some embodiments, optionally, the pirfenidone topical external preparation further comprises a humectant, which may be a humectant known in the art for topical external preparations, including but not limited to: one, two or more of glycerol, urea, allantoin, sodium hyaluronate, polyethylene glycol, squalane, and cyclomethicone. Preferably, the humectant is selected from one, two or three of glycerin, allantoin and sodium hyaluronate.

In some embodiments, the humectant is present in an amount of 0.5wt% to 10wt%, preferably 2wt% to 8wt%, for example 3wt% to 6wt%, based on the total weight of the pirfenidone topical external preparation.

In some embodiments, optionally, the pirfenidone topical external preparation comprises a pH adjustor, which may be a pH adjustor for topical external preparations known in the art, in particular an alkaline pH adjustor, such as one, two or three selected from triethanolamine, sodium hydroxide, triethylamine; preferably, the pH adjuster is triethanolamine. The pH adjustor is used in an appropriate amount, and preferably the pH of the topical preparation is adjusted to a range of 5.0 to 7.5, more preferably to a pH of 5.5 to 7.5.

In some embodiments, the pirfenidone topical external preparation optionally further comprises a preservative, which may be a preservative known in the art for topical external preparations, for example, one, two or more selected from methyl parahydroxybenzoate (methylparaben), propyl parahydroxybenzoate (propylparaben), benzyl alcohol, benzoic acid, phenol.

In some embodiments, the preservative is present in an amount of 0.01wt% to 2wt%, such as 0.05wt% to 1wt%, based on the total weight of the pirfenidone topical external preparation.

In some embodiments, optionally, the topical external formulations of pirfenidone of the present invention further include one, two or more pharmaceutically acceptable additional excipients including, but not limited to: antioxidants, thickeners, stabilizers, chelating agents, oily materials, emulsifiers, permeation enhancers, antimicrobial agents, opacifiers, fragrances, colorants, surfactants, and the like. The adjuvants may be used in conventional amounts known in the art.

For example, the antioxidant may be an antioxidant known in the art for topical preparations, such as one, two or more selected from Butyl Hydroxy Anisole (BHA), butylated hydroxy toluene, vitamin C, vitamin E, vitamin a, lutein, lycopene, retinyl palmitate, potassium metabisulfite, sodium thiosulfate pentahydrate, 3, 4-dihydroxybenzoic acid, propyl gallate, alpha-lipoic acid, ascorbyl palmitate, sodium metabisulfite, ubiquinone, selenium. For example, the antioxidant is Butyl Hydroxy Anisole (BHA). The antioxidant may be present in an amount of 0.05wt% to 5wt%, for example 0.1wt% to 3wt%, based on the total weight of the pirfenidone topical external preparation.

For example, the thickener may be, for example, one, two or more of cellulose derivatives, polyvinylpyrrolidone, carbomer polymers, carbomer derivatives, maltodextrin, polydextrose, dextrates, carboxypolymethylenes, polyvinyl alcohol, poloxamers, polyethylene glycols; more preferably, the thickener is hydroxypropyl cellulose. The thickener may be present in an amount of 0.05 to 5wt%, preferably 0.1 to 4wt%, for example 1 to 3wt%, based on the total weight of the pirfenidone topical external preparation.

In an exemplary embodiment, the topical pirfenidone formulation of the present invention comprises a therapeutically effective amount of pirfenidone, water, and at least one co-solvent comprising DMSO and ethanol, wherein the formulation comprises 4wt% to 12wt% pirfenidone, 15wt% to 30wt% DMSO, 15wt% to 30wt% ethanol, based on the total weight of the topical formulation. Optionally, the formulation further comprises 0.05wt% to 5wt% of a gelling agent (e.g., one or both selected from carbomer 980, carbomer 981). Optionally, the formulation further comprises 0.05wt% to 8wt% of a humectant (e.g., one, two or three selected from glycerol, allantoin, sodium hyaluronate). Optionally, the formulation further comprises 0.05wt% to 2wt% preservative. Optionally, the formulation further comprises a pH adjuster (e.g., one or both selected from triethanolamine, naOH) to adjust the pH of the formulation to a range of 5.0 to 7.5. Optionally, the formulation further comprises pharmaceutically acceptable additional excipients.

In an exemplary embodiment, the topical pirfenidone formulation of the present invention comprises a therapeutically effective amount of pirfenidone, water, and at least one co-solvent comprising DMSO and ethanol, wherein the formulation comprises 6wt% to 12wt% pirfenidone, 20wt% to 30wt% DMSO, 20wt% to 30wt% ethanol, based on the total weight of the topical formulation. Optionally, the formulation further comprises 0.4wt% to 1wt% of a gelling agent (e.g., one or both selected from carbomer 980, carbomer 981). Optionally, the formulation further comprises 3wt% to 6wt% of a humectant (e.g., one, two or three selected from glycerol, allantoin, sodium hyaluronate). Optionally, the formulation further comprises 0.05wt% to 0.5wt% preservative. Optionally, the formulation further comprises a pH adjuster (e.g., one or both selected from triethanolamine, naOH) to adjust the pH of the formulation to a range of 5.0 to 7.5. Optionally, the formulation further comprises pharmaceutically acceptable additional excipients.

In one exemplary embodiment, the formulation comprises 4wt% to 12wt% pirfenidone, 15wt% to 35wt% DMSO, 15wt% to 30wt% ethanol, 20wt% to 50wt% water, 0.3wt% to 1wt% gellant, 2wt% to 8wt% humectant, and an appropriate amount of pH adjuster to adjust the pH of the formulation to a range of 5.0 to 7.5.

In one exemplary embodiment, the formulation comprises 4wt% to 12wt% pirfenidone, 20wt% to 25wt% DMSO, 20wt% to 25wt% ethanol, 30wt% to 45wt% water, 0.3wt% to 1wt% gellant, 2wt% to 8wt% humectant, 0.05wt% to 1wt% preservative, and an appropriate amount of pH adjuster to adjust the pH of the formulation to a range of 5.0 to 7.5.

In a second aspect, the invention also provides the use of the topical pirfenidone preparation in the manufacture of a medicament for preventing or treating diseases associated with hyperproliferation of skin or connective tissue and chronic skin lesions.

In a third aspect, the present invention provides a method for preventing or treating chronic skin injury, a disease associated with hyperproliferation of skin or connective tissue in a mammal (e.g., a human), said method comprising administering to the mammal (e.g., a human) a therapeutically effective amount of a topical external preparation of pirfenidone according to the present invention.

In some embodiments of the invention, the skin or connective tissue hyperproliferative related disorders include, but are not limited to, pathological scars (e.g., keloids, hypertrophic scars), acne scars, skin neoplastic fibrosis, and scarring alopecia; the chronic skin lesions include, but are not limited to, vasculitis, burn healing, diabetic foot syndrome, vascular ulcers, scleroderma, peyronie's disease, du Yishi syndrome, or adhesive capsulitis.

The inventor of the invention surprisingly found that when DMSO and an alcohol solvent are used as cosolvent in a pirfenidone topical external preparation, the medicinal performance of the pirfenidone topical external preparation can be obviously improved, and the obtained preparation overcomes the technical problems of low solubility of pirfenidone in water, low skin transmittance, sensitivity to skin irritation and the like. The preparation has high skin permeability, good stability, no irritation, good skin tolerance and convenient use, can remarkably improve the compliance of patients with keloids, pathological scars or hypertrophic scars, and can be developed into a very promising topical medicine.

Drawings

Fig. 1 to 4 are graphs of HE-stained pathological sections of four different groups of rabbits after 28 days of administration in the in vivo drug efficacy test of pirfenidone in test example 4, wherein fig. 1, 2, 3 and 4 show the HE-stained pathological sections of 4 samples in each group.

Definition and description of terms

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications referred to herein are incorporated by reference in their entirety.

The terms "comprising," "including," "having," "containing," or "involving," and other variations thereof herein, are inclusive or open-ended and do not exclude additional unrecited elements or method steps.

The term "preventing or treating" means that the formulation of the invention is administered to prevent, ameliorate or eliminate a disease or one or more symptoms associated with the disease, and includes: (i) Preventing the occurrence of a disease or disease state in a mammal, particularly when such mammal is susceptible to the disease state, but has not been diagnosed as having the disease state; (ii) inhibiting the disease or disease state, i.e., inhibiting its progression; (iii) The disease or condition is alleviated, even if the disease or condition subsides.

The term "therapeutically effective amount" means an amount of a formulation of the invention that (i) treats or prevents a particular disease, condition, or disorder, (ii) alleviates, ameliorates, or eliminates one or more symptoms of a particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of a particular disease, condition, or disorder described herein. The amount of the formulation of the present invention that constitutes a "therapeutically effective amount" will vary depending on the formulation, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by one of ordinary skill in the art based on his own knowledge and disclosure.

The term "pharmaceutically acceptable" is intended to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The term "pharmaceutically acceptable excipients" refers to those excipients which do not significantly stimulate the organism and which do not impair the biological activity and properties of the active compound. Suitable excipients are well known to the person skilled in the art, such as carbohydrates, waxes, water soluble and/or water swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, water, other solvents and the like.

As used herein, the term "solvent" is water.

As used herein, the term "co-solvent" refers to a substance that acts as an adjunct to other substances, such as solvent water.

As used herein, the term "chronic skin injury" refers to the vigorous activity that results in the formation of abnormal fibrotic tissue (e.g., scarring) during various stages of wound healing.

As used herein, the term "diabetic foot syndrome" diabetic foot is a clinical entity, which refers to a syndrome resulting from the interaction of evoked factors such as vascular disease, neuropathy, and infection, caused by the presence of chronic hyperglycemia in diabetic patients, external factors and intrinsic factors such as trauma, local hygiene, and skeletal deformity. Typical clinical symptoms are ulcers of the lower extremities, foot ulcers (also known as diabetic foot ulcers) and the like of patients.

As used herein, the term "polyacrylamide-based gellant" refers primarily to a gel made by the company SEPPIC under the name Sepineo (or Simulgel 600->) The sold mixture sodium acryloyldimethyl taurate copolymer/isohexadecane/polysorbate 80, the mixture polyacrylamide/isoparaffin C13-14/laureth-7 (as named @ by SEPPIC company> 305.

Detailed Description

The technical scheme of the invention is further described in detail below with reference to specific embodiments. It should be understood that the following examples are illustrative only and are not to be construed as limiting the scope of the invention. All techniques implemented based on the above description of the invention are intended to be included within the scope of the invention. The experimental procedure, in which specific conditions are not specified in the examples, is generally carried out according to conventional conditions, or according to the conditions recommended by the manufacturer. The percentages and parts used in the examples are by weight unless otherwise indicated, and the starting materials and reagents used in the examples below are commercially available or may be prepared by known methods.

The invention adopts the following abbreviations: DMSO represents dimethyl sulfoxide; NMP represents N-methylpyrrolidone; solutol HS 15 represents polyethylene glycol (PEG) -15 hydroxystearate; transcutol stands for diethylene glycol monoethyl ether; HPC stands for hydroxypropyl cellulose, and BHA stands for butyl hydroxy anisole. In the following table "-" means that the corresponding component is not contained or added.

Example 1 solubility test of pirfenidone

This example examined the solubility of pirfenidone in water/DMSO/alcohol solvents.

The experimental method comprises the following steps: excess pirfenidone was added to the solvent system listed in table 1. The resulting suspension was stirred overnight and filtered through a 0.22 μm filter. The filtrate was diluted to the appropriate concentration. The concentration of pirfenidone in each solution was determined by HPLC method. The results of the solubility of pirfenidone in different solvent systems are shown in table 1.

TABLE 1 solubility of pirfenidone in different solvent systems

As can be seen from the experimental results, although purified water is a common solvent in topical preparations, pirfenidone has a low solubility in water, and even if solubilization is performed by adding DMSO, the solubility is difficult to reach the concentration (80 mg/mL) desired in topical preparations. However, if ethanol or propylene glycol is further added for solubilization, the solubility of pirfenidone in water can be significantly improved. For example, pirfenidone has better solubility in either water/DMSO/ethanol or water/DMSO/propylene glycol, and can reach the desired concentration of pirfenidone in topical external formulations (80 mg/mL).

Example 2 experiments on compatibility of raw materials and auxiliary materials of topical pirfenidone preparation

In the embodiment, a raw and auxiliary material compatibility experiment is carried out to examine the influence of auxiliary materials such as a cosolvent, a gelling agent, a humectant and the like on the stability of the pirfenidone topical external preparation. Gellants (e.g., carbomers) and humectants (e.g., glycerin, allantoin, sodium hyaluronate) are common adjuvants in topical external preparations.

The experimental method comprises the following steps: pirfenidone and auxiliary materials (including cosolvent, gelatinizer, humectant, etc.) are mixed in a weight ratio of 1:99 (the weight of API pirfenidone is 1% and the total weight of other auxiliary materials is 99%), and the mixture is placed for 30 days under the condition of high temperature (60 ℃ C.) and then the total degradation impurity content and the maximum single impurity content are tested. The experimental results are shown in table 2.

TABLE 2 compatibility experiments of raw materials and auxiliary materials

Experimental results show that the pirfenidone has good stability in the cosolvent (DMSO, ethanol), the common gelling agent (carbomer) and the humectant (glycerin, allantoin and sodium hyaluronate) tested in the example 1, so that the auxiliary materials have good compatibility with the pirfenidone, and can be used in the pirfenidone topical external preparation.

In addition, the stability of pirfenidone in the presence of Transcuto was also tested in the present application. Although Transcutol can also solubilize pirfenidone in dmso+water systems, use of Transcutol results in product instability. For example, a formulation containing 1% pirfenidone/Transcutol/water 23.5% 75.5% will produce significant degradation impurities after 30 days of exposure to high temperature (60℃., dry) conditions, presumably to induce degradation of pirfenidone.

In summary, it is clear that not any organic solvent can be used as a compatibilizer in the topical external formulations of pirfenidone of the present application. The DMSO+alcohol solvent used in the invention not only can solubilize, but also can not influence stability and the like of pirfenidone, thus being a good cosolvent.

Example 3 preparation of pirfenidone topical external preparation

In this example, topical external preparations of pirfenidone were prepared according to the formulation shown in table 3.

The preparation method comprises the following steps: according to the prescription, 1) pirfenidone is mixed with a cosolvent; 2) Mixing a gelling agent with a suitable amount of purified water; 3) Mixing the humectant with the rest purified water; 4) And (3) uniformly mixing the mixtures obtained in the steps (1), 2) and 3), adding a pH regulator to adjust the pH to 5.5-7.5, and stirring to uniformity to obtain the product.

TABLE 3 topical external formulations of pirfenidone

Example 4 preparation of pirfenidone topical external preparation

In this example, topical external preparations of pirfenidone were prepared according to the formulation shown in table 4.

The preparation method comprises the following steps: according to the prescription, 1) pirfenidone is mixed with cosolvent, and preservative is added for uniform mixing; 2) Mixing a gelling agent with a suitable amount of purified water; 3) Mixing the humectant with the rest purified water; 4) And (3) uniformly mixing the mixtures obtained in the steps (1), 2) and 3), adding a pH regulator to adjust the pH to 5.5-7.5, and stirring to uniformity to obtain the product.

TABLE 4 topical external formulations of pirfenidone

EXAMPLE 5 preparation of pirfenidone topical external preparation

In this example, topical external preparations of pirfenidone were prepared according to the formulation shown in table 5.

The preparation method comprises the following steps: reference example 4.

TABLE 5 topical external preparations of pirfenidone

EXAMPLE 6 preparation of pirfenidone topical external preparation

In this example, topical external preparations of pirfenidone were prepared according to the formulation shown in table 6.

The preparation method comprises the following steps: according to the prescription, 1) pirfenidone is mixed with cosolvent, and antioxidant is added for uniform mixing; 2) Mixing a gelling agent with purified water; 3) And (3) uniformly mixing the mixture obtained in the step (1) and the step (2), adding a pH regulator to adjust the pH to 5.5-7.5, and stirring to uniformity to obtain the product.

TABLE 6 pirfenidone topical external preparation

EXAMPLE 7 preparation of pirfenidone topical external preparation

In this example, topical external preparations of pirfenidone were prepared according to the formulation shown in table 7.

The preparation method comprises the following steps: reference example 3.

TABLE 7 pirfenidone topical external preparation

Example 8 preparation of pirfenidone topical external preparation

In this example, topical external preparations of pirfenidone were prepared according to the formulation shown in table 8. Wherein control formulation C1 was prepared with reference to the formulation of the pirfenidone gel formulation KitosCell marketed in mexico.

The preparation method comprises the following steps: according to the prescription, 1) pirfenidone is mixed with cosolvent, and auxiliary materials such as preservative (if any) and the like are added for mixing; 2) Mixing a gelling agent with a suitable amount of purified water; 3) And (3) uniformly mixing the mixture obtained in the step (1) and the step (2), adding a pH regulator to adjust the pH to 5.5-7.5, and stirring to uniformity to obtain the product.

TABLE 8 pirfenidone topical external preparation

Test example 1 stability of pirfenidone topical external preparation

The stability of the topical pirfenidone formulations was examined in this test example.

The test method comprises the following steps: the external preparations for pirfenidone local use in examples 3 to 8 were subjected to stability study experiments under conditions of high temperature (60 ℃, drying) and acceleration (40±2 ℃, 75% rh±5% rh), and physical and chemical stability of each preparation was examined at 0 day and 30 days. The experimental results are shown in table 9 below.

TABLE 9 stability study of pirfenidone topical external formulations

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Note that: the measurement data of day 0 is the measurement value obtained by the measurement after the completion of the sample preparation.

Experimental results show that the pirfenidone local external preparation provided by the invention is placed for 30 days under high temperature and acceleration conditions, the color properties are not changed, the impurity content is not obviously increased, and good stability is maintained. The viscosity of different formulations is greatly different due to the viscosity of the gelatinizer. For example, carbomer 980 at a concentration of 0.5% w/w has a viscosity of 40000 to 60000 centipoise at pH7.5, a viscous character, and a weak flow; whereas 0.5% w/w carbomer 981 has a viscosity of 4000 to 10000 cps at ph7.5 and a relatively high fluidity, topical gel-like formulations prepared using different gelling agents have different viscosities. When the preparation is applied, different viscosities can generate different feelings, and the preparation can be prepared without affecting the stability and the drug effect of the preparation. In addition, the results show that the addition of the humectant, the preservative, the antioxidant and the like of the invention does not affect the stability of the preparation, and is consistent with the compatibility test results of the raw materials and the auxiliary materials of the example 2.

In the formulation of the present invention, the cosolvent may be a combination of DMSO and an alcoholic solvent. In contrast, the gel formulation in control formulation C1 with polyethylene glycol glycerol hydroxystearate and NMP as solubilizers or cosolvents darkened in the formulation when left at 60 ℃ for 30 days with an increase in total degradation products to 0.34% and poor formulation stability, which may be the effect of NMP and the preservatives diazolidinyl imidazole urea, iodopropynyl butylcarbamate on the stability of the formulation; in the control prescription C2, the hydroxystearic acid polyethylene glycol glyceride and the propylene glycol are used as the cosolvent, and the stability is better than that of the control prescription C1; in the control formula C3, transcutol and DMSO are used as cosolvents, the temperature is high at 60 ℃ for 30 days, the total degradation impurities are increased to 0.60%, the Transcutol is presumed to have an influence on the stability of the preparation, and the result is consistent with the raw and auxiliary material compatibility experimental result in the example 2. In conclusion, the pirfenidone topical external preparation has better physical and chemical stability.

Test example 2 in vitro transdermal test of pirfenidone topical external preparation

In vitro transdermal tests were performed on the topical external preparations of pirfenidone in examples 3 to 8, and the ability of each topical external preparation of pirfenidone to penetrate the skin layer was evaluated. In general, hairless guinea pigs or pigs are a recognized model for predicting human skin absorption. In contrast, other models using experimental animals with hair (such as rats, rabbits and guinea pigs) often lead to overestimated human skin absorption.

The test method comprises the following steps: skin permeation/retention tests were performed using a vertical diffusion cell using the back skin of Bama pig (1.5 month old, lin.county, shangde farm products Co., ltd., no. 20220454, 20220455, 20220456) as the permeation membrane. Collecting pigskin with thickness of 0.8-1.0mm, and cutting off hair on skin. The resistance of the skin was confirmed to ensure skin integrity. The skin was placed on top of a diffusion cell in contact with the receiver, the receiving phase of which was filled with Phosphate Buffer (PBS) (pH 6.8). About 25mg of pirfenidone topical preparation was added to the skin surface in the donor compartment, after 24 hours the receiving phase medium was taken, filtered through a 0.45 μm filter, analyzed by High Performance Liquid Chromatography (HPLC), and the average cumulative permeation of the formulation was determined and calculated, the results are shown in table 10.

TABLE 10 24h cumulative permeation for different formulations

Prescription of prescription	Average cumulative permeation amount [ mu ] g/cm ²
		1	1312.7
2	1312.9
		3	1328.7
4	1439.9
		5	946.5
6	849.7
		7	1003.5
8	839.7
		9	801.7
10	932.5
		11	181.5
12	166.3
		13	1200.1
14	1189.4
		15	1201.4
16	869.3
		17	1055.6
18	989.6
		19	1278.5
20	1032.0
		C1	405.7
C2	173.0
		C3	490.0

The experimental results show that the 24h cumulative permeation of the control formula C1 is 405.7 mug/cm ² The 24h cumulative permeation of control formulation C2 was also only 173.0 μg/cm ² The 24h cumulative osmotic amount of control formulation C3 was higher than that of control formulations C1 and C2. The cumulative penetration of the pirfenidone topical external preparation on the skin for 24 hours is about 800-1300 mug/cm ² About 2-7 times that of the control recipe. In summary, the formulations of the present invention have higher skin cumulative penetration, which may be the result of DMSO and ethanol and propylene glycol as co-solvents to facilitate penetration of the pharmaceutically active ingredient. Furthermore, the experimental results show that the addition of humectants, preservatives and antioxidants does not substantially affect the cumulative permeability of the formulation.

Test example 3 skin irritation test

In addition to the above-mentioned penetration amounts, the irritation of topical preparations is also an important consideration. The test example is used for examining the skin irritation of the pirfenidone local external preparation through an acute skin irritation experiment (namely, a one-time smearing experiment).

The test method comprises the following steps: 15 volunteers were selected as subjects and the different formulation prescriptions shown in table 14 were tried out sequentially. After the back and the inner side of the arm of the subject are cleaned, an area of about 2cmx2cm (without thick heavy body hair coverage) is selected, about 0.1g of the sample is smeared in the selected area, and the sample is smoothed to a thin layer, and skin irritation scoring is performed within 2-3 min. The higher skin irritation score indicates more irritation of the corresponding formulation.

The scoring method comprises the following steps: the following scoring method was set with reference to the skin irritation/corrosiveness test method of cosmetic safety technical Specification (2015 edition). Pain scores were calculated according to equation 1[ skin pain score = (Σhandpain score x 0.4+ Σarmmedial pain score x 0.6)/number of subjects ], and pain scores were performed according to the skin pain response score criteria of table 11; the sample with pain feeling is smeared for 2-3 minutes and then immediately wiped off the test agent, the sample without obvious stinging is wiped off after waiting for 30 minutes, and skin irritation reaction is observed after wiping off the test agent; and calculating skin irritation integral according to formula 2[ skin irritation integral= (Σhand erythema and edema integral x 0.4+ Σhand inside erythema and edema integral x 0.6)/number of subjects ], skin irritation score according to skin irritation response score criteria of table 12; and the skin irritation intensity was evaluated according to table 13 by calculating the skin irritation score according to formula 3 (skin irritation score = skin pain score + skin irritation score). The experimental results are shown in Table 14.

TABLE 11 skin pain response scoring criteria

Pain response	Score (score)
		Painless	0
Slight pain	1
		Moderate pain	2
Severe pain	3

TABLE 12 skin irritation response scoring criteria

TABLE 13 skin irritation intensity evaluation Table

TABLE 14 evaluation results of skin irritation for different formulations

Prescription of prescription	Integral of skin pain (minute)	Skin irritation integral (minute)	Skin irritation score (score)	Skin irritation intensity
					4	0	0.4	0.4	No irritation
5	0	0	0	No irritation
					6	0	0	0	No irritation
9	0	0.15	0.15	No irritation
					13	0	0.15	0.15	No irritation
19	0	0.38	0.38	No irritation
					16	0	0	0	No irritation
C1	1	1.6	2.6	Light irritation

Experimental results showed that the subjects had a significantly mild pain sensation during the administration of the control formulation C1 and a significantly less irritating, which may be caused by the greater amount of NMP; whereas the formulation of the present invention does not contain NMP, but rather uses an appropriate amount of DMSO with ethanol and/or propylene glycol as a co-solvent, the subject is substantially free of pain and irritation after administration, with the administration experience of formulas 5, 6 and 16 being optimal. In addition, the moisturizing agent is added into the pirfenidone topical external preparation, so that the skin can be kept moist, the irritation is reduced, and the skin comfort is improved particularly when a subject needs to apply the medicine for a long time.

Test example 4 in vivo efficacy test of pirfenidone

Pathological scars often occur after deep dermis is damaged, resulting in damage to the aesthetics and function of the patient, severely affecting the quality of life of the patient. Since human scar tissue is often limited in clinical availability, most drug efficacy studies require the use of experimental animal models. The wound surface is established on the rabbit ears, and the perichondrium is removed, so that the wound surface can shrink slowly and heal slowly, and finally the scar is caused. The model is often used for in vivo efficacy study of scar drugs. The experiment is to apply external preparation to rabbit ear with wound surface to treat scar and observe the healing of scar through HE pathological section.

Test materials: experimental formulation 4, wherein group G1: formulation prescription 6 of the present invention; group G2: the matrix of formulation 6 of the present invention is free of pirfenidone; group G3: control recipe C1; group G4: model group, no formulation applied).

Test instrument: dehydrator (DIAPATH, donatello), embedding machine (JW JECLIPSE E100, JW JECL), freezing station (JW JECL 5), pathology slicer (Shanghai Cuff instruments Co., ltd., RM 2016), tissue film spreading machine (Pachy Korea instruments Co., jin Huashi, zhejiang), oven (Tianjin Lai Bori instruments Co., ltd., GFL-230), anti-drop slide (Servicebio), front-mounted optical microscope (NIKON ECLIPSE 100, NIKON DS-U3), imaging system (NIKON DS-U3), xylene (national medicine Cluster chemical Co., ltd., 10023418), absolute ethyl alcohol (national medicine cluster chemical Co., 100092683), HE dye set (Servic BIO; G1003), center gum (national medicine cluster chemical Co., ltd., 10004160), new Zealand (Nanj Kagaku Korea, gd., ltd., gd.: 201XF.2015).

The test method comprises the following steps: 8 male New Zealand rabbits (1.7-2.0 kg) were randomly divided into 4 groups of 4 rabbit ears each, and after labeling, the rabbits were adaptively kept for 1 week, then anesthetized, and the rabbit ear ventral hair was shaved off with a razor and subjected to conventional sterilization. The wound surface puncher with the diameter of 1cm is used, the area measured on the abdomen side of the rabbit ears is tightly pressed after being contacted with the surface of the rabbit ears, the wound surface puncher is lightly beaten by a heavy object, and three and four circles are lightly rotated, each rabbit ear causes 4 full-layer skin wounds with the diameter of 1cm multiplied by 1cm, and each wound is spaced by about 1.5cm. The skin in the wound area is removed along with the underlying cartilage membrane, retaining the cartilage. The wound was sterilized by iodophor and dosing was started the next day in the manner shown in table 15 below.

TABLE 15

Group of	Number of samples	Administration mode	Administration volume (ml)	Number of administrations per day	Days of administration
						G1	4	Local application	0.1	3	28
G2	4	Local application	0.1	3	28
						G3	4	Local application	0.1	3	28
G4	4	No application of any substance	0.1	3	28

After each administration, the medicine should be applied to the administration site of rabbit ears, and before each administration, a cotton swab should be used to dip in physiological saline to wipe the administration site. Rabbits were euthanized 28 days after dosing. The scar and normal tissue within 3mm around are cut off along with the cartilage. Meanwhile, a normal rabbit ear tissue is selected as a blank control. One marked wound per rabbit ear was fixed with 10% formalin and sectioned and HE stained.

The slicing steps are as follows: fresh tissue is fixed for more than 24 hours by using a fixing liquid. And taking out the tissue from the fixing solution, trimming the tissue of the target part in a fume hood by using a surgical knife, and placing the trimmed tissue and a corresponding label in a dehydration box. The dehydration box is put into a dehydrator to be dehydrated in a gradient way in sequence: 75% alcohol 4h,85% alcohol 2h,90% alcohol 2h,95% alcohol 1h, absolute ethanol I30 min, absolute ethanol II 30min, and alcohol benzene (ethanol, xylene mixed solvent, V) _Ethanol :V _{Xylene (P)} =1: 1) 5-10 min, 5-10 min of dimethylbenzene I, 5-10 min of dimethylbenzene II, 1h of paraffin wax I melting at 65 ℃, 1h of paraffin wax II melting at 65 ℃ and 1h of paraffin wax III melting at 65 ℃. Embedding the wax-soaked tissue in an embedding machine. Firstly, putting melted wax into an embedding frame, taking out tissues from a dehydration box before the wax is solidified, putting the tissues into the embedding frame according to the requirement of an embedding surface, and attaching corresponding labels. Cooling at-20deg.C, solidifying, removing the wax block from the embedding frame, and trimming. And (3) placing the trimmed wax block into a freezing table at the temperature of minus 20 ℃ for cooling, and then placing the cooled wax block into a paraffin slicer for slicing, wherein the thickness of the cooled wax block is 4 mu m. The slices float on warm water at 40 ℃ of a slice spreading machine to flatten the tissues, the glass slide drags the tissues out, and the slices are baked in a baking oven at 60 ℃. And (5) baking the water, drying the wax, baking, taking out and preserving at normal temperature for standby.

The HE dyeing steps are as follows: the slices are stained with hematoxylin dye liquor for 3-5 min, washed with running water, differentiated with differentiation liquor, washed with running water, returned to blue with blue returning liquor, and washed with running water. Sequentially dehydrating with 85% and 95% gradient alcohol for 5min, and dyeing in eosin dye solution for 5min. Finally, absolute ethyl alcohol I5 min, absolute ethyl alcohol II 5min, absolute ethyl alcohol III 5min, dimethylbenzene I5 min and dimethylbenzene II 5min are sequentially put into the gel sealing piece for microscope examination and image acquisition analysis.

And evaluating the tissue damage condition through pathological sections, and carrying out pathological analysis on scar tissues. And observing and scoring the conditions of fibroblasts, microvessels, collagen fibers and the like under a microscope, wherein the scoring standard is as follows:

1-3 minutes, no-minimal cell aggregation. No granulation tissue or epithelium is transited;

4-6 minutes, thin, immature granulation tissue, with inflammatory cells as the main, small amounts of fibroblasts, capillaries or collagen deposition. Minimal epithelial cell migration;

the medium thickness granulation tissue is changed from inflammatory cells to fibroblasts and collagen deposition at 7-9 min. A wide range of new blood vessels. Moderate epithelialization migration;

10-12 minutes, thicker vascular granulation tissue, with fibroblast and extensive collagen deposition being the dominant. The epithelium partially or completely covers the wound surface. According to this criterion, 16 pathological sections were scored as follows in table 16. The HE staining pathological section diagrams of four different groups of rabbits after 28 days of administration of the ear surface scar regions are respectively shown in figures 1-4.

Table 16 he pathological section scoring

Experimental results show that the pirfenidone topical external preparation group (G1) has the most obvious fiber repairing effect, and the control formula C1 (G3) is adopted, so that the scores of the blank matrix group (G2) and the model group (G4) are not different. The pirfenidone topical external preparation provided by the invention has very good treatment effect on scars, and the effect is better than that of a control prescription C1 (G3).

Test example 5 pharmacokinetic Property Studies

The test example aims at researching a single local application of pirfenidone topical external preparation to a miniature pig, detecting the concentration of active ingredients in plasma, and evaluating the in vivo Pharmacokinetic (PK) characteristics, systemic exposure and safety of the miniature pig after local administration.

Experimental materials: male Bama miniature pig (Bama Miniature Swine) (3-5 months old, weight 9-10kg, purchased from Shanghai Jia Su Biotechnology Co., ltd.), experimental formulation (formulation of the present invention, formulation No. 6).

The experimental method comprises the following steps: on the day before the experiment, 2 piglets fasted overnight. On the day of the experiment, after weighing the body weight and calculating the administration area, selecting the area marked with the corresponding area on the back of the miniature pig according to 10% of the body surface area, removing hair, cleaning, and controlling the administration amount to be 20mg/cm ² The preparations are respectively absorbed and evenly smeared for administration, and gauze covers the administration area and is fixed by waistcoat.

Blood samples were collected at 0min before administration and at 15min, 30min, 1h, 2h, 3h, 4h, 6h, 8h, 10h and 24h after administration to K ₂ EDTA anticoagulation tube, placing on wet ice, centrifuging (1500-160 g,4 ℃) for 10min within 1h, separating plasma, storing the obtained plasma sample in-90-60 ℃ environment, and placing under dry ice condition for transportation.

The drug concentration in the plasma of the minipig was detected by LC-MS/MS bioanalytical method, and the pharmacokinetic profile in the minipig was evaluated by analyzing the blood concentration-time data using a non-compartmental model using winnonlin (version 8.3, certara, USA). Table 17 shows pharmacokinetic parameters of pirfenidone in male mini-pig plasma after topical application of topical external preparations of pirfenidone.

TABLE 17 pharmacokinetic parameters of pirfenidone topical external formulations

Note that: NR indicates that it cannot be calculated.

Experimental results show that the concentration of the active components is 20mg/cm ² Is applied to male pig plasma after topical application of pirfenidone topical external preparation _max ) The AUC (which may be used to represent exposure) was 2.695h μg/mL at 150ng/mL, demonstrating that the pirfenidone topical external preparation of the present invention has good safety for long-term use.

The features and advantages of the present invention will be better understood, and appreciated, by those skilled in the art from the foregoing examples and test examples. However, the technical solution of the present invention is not limited to the above embodiments, and any modification, equivalent replacement, improvement, etc. of the formulation recipe, especially the cosolvent system, are included in the protection scope of the present invention. .

Claims

1. A topical external preparation of pirfenidone, comprising a therapeutically effective amount of pirfenidone, water, and at least one co-solvent selected from the group consisting of Dimethylsulfoxide (DMSO) and alcoholic solvents.

2. The formulation of claim 1, wherein the alcoholic solvent is selected from one, two or more of ethanol, propylene glycol, isopropanol, 1-propanol, butylene glycol.

Preferably, the cosolvent is a combination of DMSO and ethanol, or a combination of DMSO and propylene glycol, or a combination of DMSO, ethanol and propylene glycol.

3. A formulation according to claim 2, wherein the amount of water in the formulation is from 10wt% to 55wt%, preferably from 15wt% to 50wt%.

4. A formulation according to any one of claims 1 to 3, wherein when the co-solvent is a combination of DMSO and ethanol, the formulation comprises 15wt% to 35wt% DMSO, 15wt% to 30wt% ethanol, based on the total weight of the topical formulation; preferably, the formulation comprises 15wt% to 25wt% DMSO, 15wt% to 25wt% ethanol; or alternatively

When the cosolvent is a combination of DMSO, ethanol, and propylene glycol, the formulation comprises 15wt% to 35wt% DMSO, 15wt% to 30wt% ethanol, 15wt% to 25wt% propylene glycol, based on the total weight of the topical formulation; preferably, the formulation comprises 15wt% to 25wt% DMSO, 15wt% to 25% ethanol, and 20wt% to 25wt% propylene glycol.

5. The formulation of any one of claims 1-4, wherein pirfenidone is present in an amount of 1wt% to 14wt%, based on the total weight of the topical external formulation of pirfenidone.

6. The formulation of any one of claims 1-5, wherein the formulation further comprises a pharmaceutically acceptable additional adjuvant selected from one, two or more of a gelling agent, a humectant, a preservative, an antioxidant, a pH adjusting agent, a thickener, a stabilizer, a chelating agent, an oily material, an emulsifier, a penetration enhancing agent, an antimicrobial agent, an opacifying agent, a fragrance, a colorant, a surfactant.

7. The formulation of claim 1, wherein the formulation is in a dosage form selected from the group consisting of solutions, suspensions, creams, ointments, lotions, and gels.

8. The formulation of any one of claims 1-7, wherein the formulation comprises 4wt% to 12wt% pirfenidone, 20wt% to 35wt% DMSO, 20wt% to 25wt% ethanol, 20wt% to 50wt% water, 0.3wt% to 1wt% gellant, 2wt% to 8wt% humectant, and an appropriate amount of pH adjuster to adjust the pH of the formulation to a range of 5.0 to 7.5.

9. The formulation of any one of claims 1-8, wherein the formulation comprises 4wt% to 12wt% pirfenidone, 20wt% to 25wt% DMSO, 20wt% to 25wt% ethanol, 30wt% to 45wt% water, 0.3wt% to 1wt% gellant, 2wt% to 8wt% humectant, 0.05wt% to 1wt% preservative, and an appropriate amount of pH regulator to adjust the pH of the formulation to a range of 5.0 to 7.5.

10. Use of a topical pirfenidone formulation according to any one of claims 1-9 in the manufacture of a medicament for the prevention or treatment of diseases associated with hyperproliferation of skin or connective tissue, chronic skin lesions.